Lignocellulosic feedstock is a term commonly used to describe plant-derived biomass comprising cellulose, hemicellulose and lignin. Much attention and effort has been applied in recent years to the production of fuels and chemicals, primarily ethanol, from lignocellulosic feedstocks, such as agricultural wastes and forestry wastes, due to their low cost and wide availability. These agricultural and forestry wastes are typically burned or land-filled; thus using these lignocellulosic feedstocks for ethanol production offers an attractive alternative to disposal. Yet another advantage of these feedstocks is that the lignin byproduct, which remains after the cellulose conversion process, can be used as a fuel to power the process instead of fossil fuels. Several studies have concluded that, when the entire production and consumption cycle is taken into account, the production of ethanol from lignocellulosic feedstocks generates close to zero greenhouse gases.
The first chemical processing step for converting lignocellulosic feedstock to ethanol, or other fermentation products, involves breaking down the fibrous lignocellulosic material to liberate sugar monomers from the feedstock for conversion to a fermentation product in a subsequent step of fermentation.
There are various known methods for producing fermentable sugars from lignocellulosic feedstocks, one of which involves an acid or alkali pretreatment followed by hydrolysis of cellulose with cellulase enzymes and β-glucosidase. The purpose of the pretreatment is to increase the cellulose surface area and convert the fibrous feedstock to a muddy texture, with limited conversion of the cellulose to glucose. Acid pretreatment typically hydrolyses the hemicellulose component of the feedstock to yield xylose, glucose, galactose, mannose and arabinose and this is thought to improve the accessibility of the cellulose to cellulase enzymes. The cellulase enzymes hydrolyse cellulose to cellobiose which is then hydrolysed to glucose by beta-glucosidase. Hydrolysis of the cellulose and hemicellulose can also be achieved with a single-step chemical treatment in which the lignocellulosic feedstock is contacted with a strong acid or alkali under conditions sufficient to hydrolyse both the cellulose and hemicellulose components of the feedstock to sugar monomers.
After production of a stream comprising fermentable sugar from the lignocellulosic feedstock, a solids separation may be conducted to remove lignin, followed by fermentation of the sugars to ethanol or other fermentation products. If glucose is the predominant substrate present, the fermentation is typically carried out with a Saccharomyces spp. yeast that converts this sugar and other hexose sugars present to ethanol. However, glucose can also be fermented to other commercial products including lactic acid, sorbitol, acetic acid, citric acid, ascorbic acid, propanediol, butanediol, xylitol, acetone, and butanol. This conversion can be carried out by a variety of organisms, including Saccharomyces spp.
The pentose sugars, xylose and arabinose, which arise from the hemicellulose component of the feedstock during acidic pretreatment, can be fermented to ethanol. However, a vast majority of wild-type Saccharomyces strains do not naturally contain all the genes required for converting these sugars to ethanol. Thus they must be introduced into the yeast to allow for this conversion. Recombinant yeasts that are able to convert xylose to ethanol are described, for example, in U.S. Pat. Nos. 5,789,210 and 6,475,768 and EP 1 727 890.
One problem with the fermentation of sugar to ethanol or other fermentation products is that bacteria can propagate quickly as the optimum conditions of the fermentation are also conducive to their growth. Unwanted byproducts that can be produced by bacterial contaminants during fermentation include lactic acid, acetone and propionic acid. Lactic acid is a common byproduct produced by bacteria such as Lactobacillus spp, Pediococcus spp, Leuconostoc spp and/or Weissella spp (amongst others) during ethanol fermentations. The production of such undesirable byproducts decreases the yield of the desired fermentation product as the bacteria compete with the yeast for fermentable sugars and convert them to undesirable byproducts instead of the fermentation product of interest. Moreover, organic acids and other byproducts can be inhibitory to the yeast. Each of these factors can contribute to decreases in the efficiency of the fermentation by lengthening the time required for carrying out the fermentation, increasing the amount of yeast required and/or decreasing the final yields to the desired fermentation product from the fermentable sugars.
Microbial contamination is especially problematic when the concentration of yeast in the fermentor is increased by yeast recycle. Yeast recycle is employed to improve the efficiency of fermentation processes that are subject to slow reaction kinetics relative to glucose fermentation such as those involving the conversion of xylose to ethanol or when it is beneficial to increase volumetric conversion rates. Increases in the volumetric rate of conversion of fermentable sugar to ethanol can be achieved by continuously separating yeast from the harvested fermentation broth, such as by centrifugation, and then re-circulating the yeast back to the fermentor. By re-introducing yeast into the reactor in this manner, the concentration of yeast in the fermentor is continuously maintained at a high level, without significant diversion of sugars to cell growth and away from the desired fermentation product. However, as a result of such repeated re-circulation of yeast, unwanted microbes, such as bacteria, are also recycled along with the yeast. As bacteria tend to divide more quickly than yeast, this can lead to significant levels of microbial contamination.
de Oliva-Neto and Yokoya (Brazilian Journal of Microbiology, 2001, 3:10-14) examined the effect of a variety of antimicrobial compounds on the viability of Saccharomyces cerevisiae, Lactobacillus and Leuconostoc in fermentations carried out on cane juice to produce ethanol. This included formulated chemicals, such as zinc manganese ethylenebis(dithiocarbamate), methyldithiocarbamate, 3-methyl-4-chlorophenol, 2-benzyl-4-chlorophenol and o-phenylphenol, 2-chloroacetamide and others, that are commonly recommended for use in microbial control in sugar and alcohol factories. Antibiotics tested included penicillum, clindamycin and cephamandole. The results showed that current chemical biocides used in industrial fuel alcoholic fermentations reduced yeast viability, while antibiotics were effective at reducing bacterial growth, without affecting yeast viability.
However, the use of antibiotics in fuel ethanol applications has its limitations as microbial contaminants are known to develop antibiotic resistance (Lushia and Heist, 2005, Ethanol Producer Magazine, Antibiotic-Resistant Bacteria in Fuel Ethanol Fermentations). Moreover, antibiotics can be carried through to dried distillers grain, which is a byproduct of commercial ethanol plants used in animal feeds, and this valuable byproduct cannot be sold if antibiotics are used in the process.
Bacterial control in industrial fuel alcoholic fermentation can also be carried out by sulfuric acid washing of yeast cell suspensions. Commercial fuel ethanol in Brazil is produced by fed-batch or continuous fermentation of sugar cane by Saccharomyces cerevisiae and employs yeast cell recycle (de Oliva-Neto and Yokoya, supra). The goal of the acid treatment is to destroy contaminating microorganisms that cannot withstand low pH conditions, without a substantial reduction in yeast viability or fermentative capacity.
US2009/0117633 discloses a process for producing ethanol from corn in which a combined saccharification and fermentation are conducted at pH values such as 3.5 to 4.0. The enzymes used in the saccharification are amylases that are adapted for hydrolysing starch under these relatively low pH values. The low pH saccharification/fermentation is conducted with the view of reducing bacterial contaminants such as lactic acid-producing and acetic acid-producing bacteria, which grow best at pH 5.0 and above. Thus, in the pH range of 3.0 to 4.5, it is believed that ethanol fermentation will predominate because yeast will grow better than contaminating bacteria.
The use of oxidants to control microbial contamination in ethanol fermentations is also known. For example, Chang et al. (Appl. Environ. Microbiol., 1997, 63: 1-6) disclose the use of sulfite and hydrogen peroxide to control bacterial contamination in the fermentation of malt extract to ethanol with yeast recycle.
Chlorine dioxide is an oxidant that is known to have a bacteriocidal effect and has been used as a disinfectant of drinking water and in the food and beverage industry. There are various known methods for producing chlorine dioxide, (see Alternative Disinfectants and Oxidants Guidance Manual, United States Environmental Protection Agency, April 1999, Chapter 4. Chlorine Dioxide, which is incorporated herein by reference) one of which involves reacting sodium chlorite with acid according to the following reaction:5NaClO2+4H+4ClO2+4Na++Na+Cl−+2H2O.Sodium chlorite is often referred to as “stabilized chlorine dioxide” or “SCD”.
The use of chlorine dioxide in ethanol fermentations is known as set forth in WO 2007/097874, WO 2009/026706, WO 2007/149450 and Johnson and Kunz (The New Brewer, 1998, Coming Clean—A New Method of Washing Yeast Using Chlorine Dioxide Vol. 15 #5-P56). WO 2007/097874 discloses a process in which chlorine dioxide is added to a fermentation tank, to a fermentable carbohydrate added to a fermentation tank, or to a propagation or conditioning system used to prepare the inoculum for a fermentation. WO 2009/026706 discloses the use of chlorine dioxide to reduce bacterial contamination in a fermentation process employing yeast recycle and utilizing sugars from lignocellulosic feedstocks. The chlorine dioxide was used to treat a yeast slurry separated from the fermentation prior to its reintroduction to the fermentor. WO 2007/149450 discloses a method for preventing the growth of bacterial contaminants in yeast fermentations to produce ethanol via the addition of stabilized chlorine dioxide. The stabilized chlorine dioxide was added prior to any significant propagation of bacteria in the system, such as to the inoculant or to fermentable sugars before their introduction to the fermentation system. As the pH of the solution is lowered due to the generation of organic acids produced by bacterial contaminants, activated chlorine dioxide is generated in situ from the stabilized chlorine dioxide and further growth of the bacteria was prevented. Johnson and Kunz (The New Brewer, 1998, Coming Clean—A New Method of Washing Yeast Using Chlorine Dioxide Vol. 15 #5-P56) discloses the use of chlorine dioxide to wash yeast during the brewing of beer as an alternative to acid washing.
The effects of ClO2 concentration on bacterial cell kill and yeast viability and fermentative capacity have been examined in ethanol fermentations (see co-owned and co-pending WO 2009/026706), but less information is available regarding the effect of other variables on chlorine dioxide efficacy, such as pH. However, the impact of pH on the effectiveness of chlorine dioxide in other industrial applications has been studied. In the beverage industry, it has been reported that chlorine dioxide has a constant efficacy at a pH level between 4 and 10, with the rate of sterilization being greater at high pH. (“Chlorine Dioxide in the Beverage Industry”, Petplanet Insider, September 2005, 6:46-47). Chlorine dioxide bleaching stages in pulp bleaching applications are conducted at acidic pH values, although there is still some controversy about the optimal pH (Reeve, 1996, Section IV: The Technology of Chemical Pulp Bleaching, Chapter 3: Chlorine Dioxide in Delignification In Pulp Bleaching, Principles and Practice, Ed. by Dence and Reeve, Tappi Press). Foegeding et al. (1986, Journal of Food Science, 51(1):197-201) assessed chlorine dioxide inactivation of Bacillus and Clostridium spores in water buffered at pH values of 4.5, 6.5 and 8.5 with phosphoric acid and it was found that C. perfuringens spores were inactivated more at pH 8.5 than at 6.5.